US11560599B2 - Onion discrimination method - Google Patents

Onion discrimination method Download PDF

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US11560599B2
US11560599B2 US16/650,677 US201816650677A US11560599B2 US 11560599 B2 US11560599 B2 US 11560599B2 US 201816650677 A US201816650677 A US 201816650677A US 11560599 B2 US11560599 B2 US 11560599B2
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nucleotide sequence
onion
seq
nucleotides
primer
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US20200248276A1 (en
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Masahiro Kato
Noriya Masamura
Masae Ueyama
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House Foods Group Inc
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/04—Stems
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/10—Processes for modifying non-agronomic quality output traits, e.g. for industrial processing; Value added, non-agronomic traits
    • A01H1/101—Processes for modifying non-agronomic quality output traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine or caffeine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/04—Amaryllidaceae, e.g. onion
    • A01H6/045—Allium cepa [onion]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y404/00—Carbon-sulfur lyases (4.4)
    • C12Y404/01—Carbon-sulfur lyases (4.4.1)
    • C12Y404/01004—Alliin lyase (4.4.1.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • a computer readable text file entitled “SequenceListing.txt,” created on or about Mar. 25, 2020 with a file size of about 9 kb contains the sequence listing for this application and is hereby incorporated by reference in its entirety.
  • the present invention relates to a method of discriminating whether an onion is an onion with no pungent taste and/or tear-inducing property, a primer, a primer set, and a kit used for the method, and a method of breeding an onion using the method.
  • Patent Literature 1 discloses that 29 types of alliinases, which are enzymes involved in production of lachrymatory factor (LF), a component causing a pungent taste, are expressed in an onion; that, among them, a specific alliinase consisting of an amino acid sequence as set forth in SEQ ID NO:5 in Patent Literature 1 is the primary alliinase associated with a pungent taste and a tear-inducing property of an onion; and that two lines of tearless and non-pungent onions were produced in which the activity of the specific alliinase was suppressed.
  • LF lachrymatory factor
  • Non Patent Literature 1 also discloses that tearless and non-pungent onions were produced in which the activity of the alliinase was suppressed, as in Patent Literature 1.
  • Non Patent Literature 2 discloses an amino acid sequence of an alliinase that has a high sequence identity to the amino acid sequence of the specific alliinase disclosed in Patent Literature 1. However, Non Patent Literature 2 does not show the magnitude of contribution of this alliinase to a pungent taste or a tear-inducing property or does not suggest the presence of other alliinases.
  • Non Patent Literatures 3 to 6 suggest the presence of onion alliinases other than the specific alliinase disclosed in Non Patent Literature 2.
  • these literatures disclose neither sequence information of these other alliinases nor explanation of whether they are associated with a pungent taste and a tear-inducing property.
  • Patent Literature 1 The onion with suppressed expression of the specific alliinase consisting of the amino acid sequence as set forth in SEQ ID NO:5 in Patent Literature 1, which is disclosed in Patent Literature 1, does not have a pungent taste or a tear-inducing property. However, Patent Literature 1 does not disclose the gene sequence of the specific alliinase or cause of not being expressed (e.g., destruction of regulatory region, transcription factor, or the gene itself). Additionally, Patent Literature 1 does not disclose information on the alliinase gene that was not expressed.
  • Non Patent Literature 1 also discloses that tearless and non-pungent onions were produced in which the activity of the alliinase was suppressed, as in Patent Literature 1, but does not disclose the complete sequence of the alliinase with suppressed expression or cause of the suppressed expression.
  • the onion alliinase gene is a multicopy gene. As disclosed in Patent Literature 1, there are 29 alliinase genes that are expressed, and even more alliinase genes are present in an onion if genes that are not expressed are included. Additionally, Non Patent Literatures 2 to 6 also indicate the presence of alliinase genes other than the specific alliinase genes disclosed in Patent Literature 1. Thus, other alliinase genes might be detected in error, and it was therefore difficult to discriminate whether the onion is an onion with no pungent taste by determining the presence or absence of the gene encoding the specific alliinase disclosed in Patent Literature 1.
  • the present invention provides a means for discriminating whether an onion is an onion with no pungent taste and/or tear-inducing property by specifically determining the presence of an alliinase gene associated with a pungent taste and a tear-inducing property in the onion in distinction from other genes.
  • a method of discriminating traits of an onion comprising:
  • the onion is discriminated to be an onion with no pungent taste and/or tear-inducing property if the presence of the nucleotide sequence of SEQ ID NO:1 is not determined in the first determination step and the presence of the nucleotide sequence of SEQ ID NO:2 is determined in the second determination step, and
  • the onion is discriminated to be an onion with a pungent taste and/or a tear-inducing property if the presence of the nucleotide sequence of SEQ ID NO:1 is determined in the first determination step, and the presence of the nucleotide sequence of SEQ ID NO:2 is determined in the second determination step.
  • the first determination step comprises determining presence of a first mutation site comprising one or more nucleotides selected from nucleotides at positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleotide sequence of SEQ ID NO:1 in the nucleic acid, and
  • the second determination step comprises determining presence of a second mutation site comprising one or more nucleotides selected from nucleotides at positions 34, 70, 1667, 1703, 127, 409, 943, and 1822 in the nucleotide sequence of SEQ ID NO:2 in the nucleic acid.
  • the first determination step comprises:
  • the second determination step comprises:
  • the first determination step comprising determining presence of a first mutation site comprising one or more nucleotides selected from nucleotides at positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleotide sequence of SEQ ID NO:1 in the nucleic acid, wherein
  • the onion is discriminated to be an onion with no pungent taste and/or tear-inducing property if the presence of the nucleotide sequence of SEQ ID NO:1 is not determined in the first determination step, and
  • the onion is discriminated to be an onion with a pungent taste and/or a tear-inducing property if the presence of the nucleotide sequence of SEQ ID NO:1 is determined in the first determination step.
  • the first determination step comprises:
  • a method of breeding an onion with no pungent taste and/or tear-inducing property comprising:
  • a primer comprising a first polynucleotide comprising a first nucleotide sequence at the 3′ end, wherein the first nucleotide sequence is identical or homologous to a first partial nucleotide sequence of 10 or more consecutive nucleotides comprised in the nucleotide sequence of SEQ ID NO:1, the first partial nucleotide sequence comprising a first nucleotide selected from positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleotide sequence of SEQ ID NO:1 within 2 nucleotides from the 3′ end.
  • a primer comprising a second polynucleotide comprising a second nucleotide sequence at the 3′ end, wherein the second nucleotide sequence is identical or homologous to a second partial nucleotide sequence of 10 or more consecutive nucleotides comprised in a complementary nucleotide sequence to the nucleotide sequence of SEQ ID NO:1, the second partial nucleotide sequence comprising a nucleotide complementary to a second nucleotide selected from positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleotide sequence of SEQ ID NO:1 within 2 nucleotides from the 3′ end.
  • a primer set comprising:
  • a primer comprising a third polynucleotide comprising a third nucleotide sequence at the 3′ end, wherein the third nucleotide sequence is identical or homologous to a third partial nucleotide sequence of 10 or more consecutive nucleotides comprised in the nucleotide sequence of SEQ ID NO:2, the third partial nucleotide sequence comprising a third nucleotide selected from positions 34, 70, 1667, 1703, 127, 409, 943, and 1822 in the nucleotide sequence of SEQ ID NO:2 within 2 nucleotides from the 3′ end.
  • a primer comprising a fourth polynucleotide comprising a fourth nucleotide sequence at the 3′ end, wherein the fourth nucleotide sequence is identical or homologous to a fourth partial nucleotide sequence of 10 or more consecutive nucleotides comprised in a complementary nucleotide sequence to the nucleotide sequence of SEQ ID NO:2, the fourth partial nucleotide sequence comprising a nucleotide complementary to a fourth nucleotide selected from positions 34, 70, 1667, 1703, 127, 409, 943, and 1822 in the nucleotide sequence of SEQ ID NO:2 within 2 nucleotides from the 3′ end.
  • a primer set comprising:
  • the third nucleotide and the fourth nucleotide are identical to each other, or the third nucleotide is positioned more closely to the 5′ end than the fourth nucleotide in the nucleotide sequence of SEQ ID NO:2.
  • a kit for discriminating traits of an onion comprising:
  • a marker gene for discriminating a pungent taste and/or a tear-inducing property of an onion for which the cDNA nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:1.
  • an onion with no pungent taste and/or tear-inducing property, which cannot be discriminated in appearance, can be discriminated accurately.
  • a target onion with no pungent taste and/or tear-inducing property can be selected accurately in a setting of breeding a new variety using an onion with no pungent taste and/or tear-inducing property as one of materials.
  • the primers, the primer sets, and the kit of the present invention can be used to amplify alliinase gene 1 or alliinase gene 2 specifically.
  • the marker gene of the present invention can be used to detect a pungent taste and/or a tear-inducing property of an onion.
  • FIG. 1 - 1 shows the alignment results for the nucleotide sequence of SEQ ID NO:1, the nucleotide sequence of SEQ ID NO:2, and the nucleotide sequence of the alliinase gene of GenBank Accession No. AAA32639.1 and the position of each primer.
  • FIG. 1 - 2 Continued from FIG. 1 - 1
  • FIG. 1 - 3 Continued from FIG. 1 - 1
  • FIG. 1 - 4 Continued from FIG. 1 - 1
  • FIG. 2 shows the detection results for amplification products of PCR on the genomic DNAs of onion #6 with no pungent taste and a control variety as templates using a primer set of the nucleotide sequences of SEQ ID NOS: 3 and 4.
  • FIG. 3 - 1 shows the detection results for alliinase gene 1 and alliinase gene 2 in amplification products of PCR on the genomic DNAs of onion #6 with no pungent taste and a control variety as templates using various primer sets.
  • FIG. 3 - 2 Continued from FIG. 3 - 1
  • FIG. 4 shows the detection results for alliinase gene 1 and alliinase gene 2 in amplification products of PCR on the genomic DNAs of 14 varieties of commercially available onions, a control variety, and onion #6 as templates using various primer sets.
  • FIG. 5 shows the detection results for alliinase gene 1 in amplification products of PCR on the genomic DNAs of leaves of a plurality of F2 onions derived from onion #6 with no pungent taste as templates using SF2-SR1 as a primer set.
  • FIG. 6 shows the measurement results for the quantity of pyruvic acid produced in onion bulbs in which alliinase gene 1 was detected in the genomic DNA (selected bulbs) and onion bulbs in which alliinase gene 1 was not detected (non-selected bulbs), among F2 onions derived from onion #6 with no pungent taste.
  • FIG. 7 - 1 shows the positions of primers in the nucleotide sequence of SEQ ID NO:1.
  • FIG. 7 - 2 Continued from FIG. 7 - 1
  • FIG. 8 - 1 shows the positions of primers in the nucleotide sequence of SEQ ID NO:2.
  • FIG. 8 - 2 Continued from FIG. 8 - 1 .
  • gene 1 refers to a gene encoding an alliinase comprised in the genomic DNA which is transcribed to an mRNA corresponding to a cDNA comprising the nucleotide sequence of SEQ ID NO:1.
  • gene 2 refers to a gene encoding an alliinase, the genomic DNA nucleotide sequence of which comprises the nucleotide sequence of SEQ ID NO:2.
  • the “gene for which the cDNA nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:1” as used herein refers to a gene for which the nucleotide sequence of the cDNA corresponding to mRNA transcribed from the gene comprises the nucleotide sequence of SEQ ID NO:1.
  • Messenger RNA refers to a mature mRNA consisting of exon regions ligated by splicing of a precursor mRNA biosynthesized by RNA polymerase from the genomic DNA as a template in the cell of an onion.
  • the process of production of mRNA (mature mRNA) from genomic DNA is “transcription.”
  • cDNA refers to a double-stranded DNA consisting of a first DNA complementary to mRNA, which is synthesized by reverse transcriptase using mRNA as a template, and a second DNA complementary to the first DNA (i.e., consisting of a nucleotide sequence obtained by substituting u with t in the nucleotide sequence of mRNA), the single-stranded first DNA, or the single-stranded second DNA.
  • the “nucleotide sequence of the cDNA corresponding to mDNA” as used herein refers to the nucleotide sequence of the second DNA.
  • polynucleotide refers to a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA), typically a DNA.
  • RNA thymine (T) can be substituted with uracil (U).
  • U uracil
  • the polynucleotide may comprise modified nucleotides, such as inosine (I), as part thereof.
  • the polynucleotide may be present as a single-stranded chain or a double-stranded chain. If the polynucleotide is present as a double-stranded chain, it is sufficient that at least one strand is a polynucleotide having characteristics detailed in this specification.
  • the method of manufacturing a polynucleotide is not particularly limited, and the polynucleotide can be manufactured using a polynucleotide synthesizer.
  • the “nucleic acid derived from an onion” refers to a genomic DNA, an mRNA, or a cDNA derived from an onion of which traits are to be discriminated (target onion).
  • the genomic DNA, the mRNA, and the cDNA also include amplified fragments of the genomic DNA, amplified fragments of the mRNA, and amplified fragments of the cDNA, respectively.
  • nucleotide sequence Y homologous to a nucleotide sequence X refers to a combination of a DNA chain consisting of the complementary sequence to the nucleotide sequence X and a DNA chain consisting of the nucleotide sequence Y that can form hydrogen bonds enough to hybridize these DNA chains under the annealing condition of a nucleic acid amplification reaction to form a stable double-stranded chain, and the nucleotide sequences X and Y may be partially different.
  • a polynucleotide consisting of the complementary sequence to the nucleotide sequence X and a polynucleotide consisting of the nucleotide sequence Y may comprise some mismatches, such as one mismatch in 10 nucleotides, one mismatch in 20 nucleotides, or one mismatch in 30 nucleotides.
  • the expression a nucleotide sequence Y “homologous” to a nucleotide sequence X means that the nucleotide sequences X and Y satisfy any of the following relationships:
  • the nucleotide sequence Y is a nucleotide sequence obtained by deleting, substituting, adding, and/or inserting one or several nucleotides in the nucleotide sequence X;
  • nucleotide sequence Y is a nucleotide sequence having a 90% or higher identity to the nucleotide sequence X;
  • a polynucleotide consisting of the nucleotide sequence Y can be hybridized with a polynucleotide consisting of the nucleotide sequence complementary to SEQ ID NO: X under stringent conditions;
  • one or several means preferably one or two, and most preferably one.
  • the value of identity indicates a value obtained using a software to calculate the identity between a plurality of nucleotide sequences (e.g., FASTA, DNASIS, and BLAST) under the default setting.
  • the value of nucleotide sequence identity is obtained by counting the number of coincident nucleotides when a pair of nucleotide sequences are aligned at the maximum degree of coincidence and calculating the proportion of the number of coincident nucleotides to the total number of nucleotides in the compared nucleotide sequence.
  • a software to calculate the identity between a plurality of nucleotide sequences e.g., FASTA, DNASIS, and BLAST
  • the value of nucleotide sequence identity is obtained by counting the number of coincident nucleotides when a pair of nucleotide sequences are aligned at the maximum degree of coincidence and calculating the proportion of the number of coincident nucleotides to the total number of nucleot
  • the identity is more preferably 95% or higher, more preferably 96% or higher, more preferably 97% or higher, more preferably 98% or higher, and more preferably 99% or higher.
  • stringent conditions means conditions under which a so-called specific hybrid is formed, and a nonspecific hybrid is not formed.
  • the stringent conditions can be appropriately determined with reference to Green and Sambrook, Molecular Cloning, 4th Ed. (2012), Cold Spring Harbor Laboratory Press. Specifically, stringent conditions can be set for temperature and a salt concentration in a solution for southern hybridization and temperature and a salt concentration in a solution for the washing process in southern hybridization.
  • the method of discriminating traits of an onion of the present invention is a method comprising
  • the onion is discriminated to be an onion with no pungent taste and/or tear-inducing property if the presence of the nucleotide sequence of SEQ ID NO:1 is not determined in the first determination step and the presence of the nucleotide sequence of SEQ ID NO:2 is determined in the second determination step, and
  • the onion is discriminated to be an onion with a pungent taste and/or a tear-inducing property if the presence of the nucleotide sequence of SEQ ID NO:1 is determined in the first determination step and the presence of the nucleotide sequence of SEQ ID NO:2 is determined in the second determination step.
  • This method is the “first discrimination method” of the present invention.
  • the present inventors found that, as the nucleotide sequence of the gene encoding alliinase, both the nucleotide sequence of gene 1 and the nucleotide sequence of gene 2 were present in the genomic DNA of a usual onion, whereas the nucleotide sequence of gene 2 was present but the nucleotide sequence of gene 1 was not present in the genomic DNA of an onion with no pungent taste and/or tear-inducing property.
  • the alliinase encoded by the nucleotide sequence of gene 1 is a specific alliinase consisting of the amino acid sequence as set forth in SEQ ID NO:5 disclosed in Patent Literature 1.
  • Patent Literature 1 discloses that an onion with suppressed expression of the specific alliinase does not have a pungent taste and/or a tear-inducing property, the cause of not being expressed (e.g., regulatory region, transcription factor, destruction of gene itself) is not disclosed.
  • the present inventors surprisingly found that the nucleotide sequence of gene 1 encoding the specific alliinase was present in the genomic DNA of an onion with no pungent taste and/or tear-inducing property, and that the nucleotide sequence of gene 2 with a high homology to the nucleotide sequence of gene 1, which is not associated with a pungent taste, was present in the genomic DNA.
  • representative examples of the “onion with no pungent taste and/or tear-inducing property” include, but are not limited to, an onion of which seed has been deposited in an International Depository Authority under Accession No. NCIMB 42219, its progenies, and onions with no pungent taste and/or tear-inducing property bred using these onions as one of materials.
  • the nucleotide sequence of SEQ ID NO:1 is the cDNA nucleotide sequence of alliinase gene 1
  • the nucleotide sequence of SEQ ID NO:2 is the genomic DNA nucleotide sequence of alliinase gene 2.
  • the “determining presence of the nucleotide sequence of SEQ ID NO:1” and “determining presence of the first mutation site (in the nucleotide sequence of SEQ ID NO:1)” in the first determination step mean “determining presence of a nucleotide sequence corresponding to the nucleotide sequence of SEQ ID NO:1” and “determining presence of a nucleotide sequence corresponding to the first mutation site (in the nucleotide sequence of SEQ ID NO:1),” respectively.
  • determining presence of the nucleotide sequence of SEQ ID NO:2” and “determining presence of the second mutation site (in the nucleotide sequence of SEQ ID NO:2)” in the second determination step mean “determining presence of a nucleotide sequence corresponding to the nucleotide sequence of SEQ ID NO:2” and “determining presence of a nucleotide sequence corresponding to the second mutation site (in the nucleotide sequence of SEQ ID NO:2),” respectively.
  • nucleotide sequence corresponding to the nucleotide sequence of SEQ ID NO:1 refers to the genomic DNA nucleotide sequence of an alliinase gene for which the cDNA nucleotide sequence is the nucleotide sequence of SEQ ID NO:1 or the complementary nucleotide sequence thereto if the nucleic acid derived from an onion is a genomic DNA; a nucleotide sequence derived from SEQ ID NO:1 by substitution of T with U if the nucleic acid derived from an onion is an mRNA; or the nucleotide sequence of SEQ ID NO:1 or the complementary nucleotide sequence thereto if the nucleic acid derived from an onion is a cDNA.
  • nucleotide sequence corresponding to the nucleotide sequence of SEQ ID NO:2 refers to the nucleotide sequence of SEQ ID NO:2 or the complementary nucleotide sequence thereto if the nucleic acid derived from an onion is a genomic DNA; a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO:2 by deletion of the intron regions and substitution of T with U if the nucleic acid derived from an onion is an mRNA; or a nucleotide sequence in which intron regions are removed from the nucleotide sequence of SEQ ID NO:2 or the complementary nucleotide sequence thereto if the nucleic acid derived from an onion is a cDNA.
  • nucleotide sequence corresponding to the first mutation site refers to the nucleotide sequence of a site corresponding to the first mutation site in the nucleotide sequence of the genomic DNA of an alliinase gene for which the cDNA nucleotide sequence is the nucleotide sequence of SEQ ID NO:1 or the complementary nucleotide sequence thereto if the nucleic acid derived from an onion is a genomic DNA; the nucleotide sequence of a site corresponding to the first mutation site in a nucleotide sequence derived from SEQ ID NO:1 by substitution of T with U if the nucleic acid derived from an onion is an mRNA; or the first mutation site in the nucleotide sequence of SEQ ID NO:1 or the complementary nucleotide sequence thereto if the nucleic acid derived from an onion is a cDNA.
  • nucleotide sequence corresponding to the second mutation site refers to the second mutation site in the nucleotide sequence of SEQ ID NO:2 or the complementary nucleotide sequence thereto if the nucleic acid derived from an onion is a genomic DNA; the nucleotide sequence of a site corresponding to the second mutation site in a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO:2 by deletion of the intron regions and substitution of T with U if the nucleic acid derived from an onion is an mRNA; or the nucleotide sequence of a site corresponding to the second mutation site in the nucleotide sequence derived from the nucleotide sequence of SEQ ID NO:2 by deletion of the intron regions, or the complementary nucleotide sequence thereto if the nucleic acid derived from an onion is a cDNA.
  • a genomic DNA is preferable to analyze a genomic DNA as a nucleic acid derived from an onion because the quantity does not change depending on the growth stage of the target onion or the site thereof. Furthermore, a genomic DNA is preferable because whether an analysis is being performed under appropriate conditions can be confirmed by confirming not only the presence or absence of the nucleotide sequence of gene 1, but also the presence of the nucleotide sequence of gene 2, which is present regardless of the presence of a pungent taste of an onion in the first discrimination method of the present invention.
  • the first determination step is a step of determining the presence of the nucleotide sequence of SEQ ID NO:1 in a nucleic acid derived from an onion.
  • a nucleotide sequence comprising at least a partial nucleotide sequence of the nucleotide sequence of SEQ ID NO:1 or of a nucleotide sequence derived from SEQ ID NO:1 by substitution of T with U or the complementary nucleotide sequence to the partial nucleotide sequence is detected in the nucleic acid derived from the onion.
  • nucleotide sequence of SEQ ID NO:1 which is the cDNA nucleotide sequence of gene 1
  • nucleotide sequence of SEQ ID NO:2 which is the genomic DNA nucleotide sequence of gene 2
  • positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleotide sequence of SEQ ID NO:1 and positions 34, 70, 1667, 1703, 127, 409, 943, and 1822 in the nucleotide sequence of SEQ ID NO:2 are mismatching nucleotides comprised in the regions with a high homology.
  • the first determination step it is preferable to determine the presence of a first mutation site comprising one or more nucleotides selected from nucleotides at positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleotide sequence of SEQ ID NO:1.
  • a first mutation site comprising one or more nucleotides selected from nucleotides at positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleotide sequence of SEQ ID NO:1.
  • a first nucleic acid amplification reaction using the following first primer set and a nucleic acid derived from an onion, in particular, a genomic DNA or a cDNA as a template and to confirm an amplification product of the first nucleic acid amplification reaction.
  • a first primer set preferably comprises at least either one of
  • a first primer comprising a polynucleotide comprising a nucleotide sequence which is identical or homologous to a partial nucleotide sequence comprising the first mutation site in the nucleotide sequence of SEQ ID NO:1, and
  • a second primer comprising a polynucleotide comprising a nucleotide sequence which is identical or homologous to the complementary nucleotide sequence to a partial nucleotide sequence comprising the first mutation site in the nucleotide sequence of SEQ ID NO:1, and particularly preferably both the first and second primers.
  • Examples of a preferred embodiment of the first primer include a first primer comprising a first polynucleotide comprising a first nucleotide sequence at the 3′ end which is identical or homologous to a first partial nucleotide sequence of 10 or more consecutive nucleotides comprised in the nucleotide sequence of SEQ ID NO:1, the first partial nucleotide sequence comprising a first nucleotide selected from positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleotide sequence of SEQ ID NO:1 within 2 nucleotides from the 3′ end.
  • Examples of a preferred embodiment of the second primer include a second primer comprising a second polynucleotide comprising a second nucleotide sequence at the 3′ end which is identical or homologous to a second partial nucleotide sequence of 10 or more consecutive nucleotides comprised in the complementary nucleotide sequence to the nucleotide sequence of SEQ ID NO:1, the second partial nucleotide sequence comprising a nucleotide complementary to a second nucleotide selected from positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleotide sequence of SEQ ID NO:1 within 2 nucleotides from the 3′ end.
  • the first partial nucleotide sequence described in association with the first primer is a partial nucleotide sequence of 10 or more consecutive nucleotides, preferably 15 or more consecutive nucleotides, more preferably 17 or more consecutive nucleotides, more preferably 20 or more consecutive nucleotides, and more preferably 23 or more consecutive nucleotides comprised in the nucleotide sequence of SEQ ID NO:1.
  • the upper limit of the number of nucleotides in the first partial nucleotide sequence is not particularly limited, and is preferably 50 or fewer nucleotides, more preferably 40 or fewer nucleotides, more preferably 35 or fewer nucleotides, and more preferably 30 or fewer nucleotides.
  • the first partial nucleotide sequence comprises the first nucleotide as a nucleotide within 2 nucleotides from the 3′ end, preferably as a nucleotide at the 3′ end, a region on the 3′ end side from a nucleotide corresponding to the first nucleotide in the nucleotide sequence on the sense chain of gene 1 in the template nucleic acid is specifically elongated from the 3′ end of the first primer in the first nucleic acid amplification reaction.
  • the first polynucleotide comprises the first nucleotide sequence which is identical or homologous to the first partial nucleotide sequence at the 3′ end.
  • the term “homologous” is defined as described above.
  • the first partial nucleotide sequence and the first nucleotide sequence are identical to each other preferably in a region of 3 nucleotides at the 3′ end, more preferably in a region of 5 nucleotides at the 3′ end, more preferably a region of 8 nucleotides at the 3′ end, and more preferably in a region of 10 nucleotides at the 3′ end (further preferably in a region of 15 nucleotides at the 3′ end if the length of the first partial nucleotide sequence is 15 or more nucleotides, further preferably in a region of 17 nucleotides at the 3′ end if the length of the first partial nucleotide sequence is 17 or more nucleotides, and further preferably
  • the first nucleotide sequence include the nucleotide sequence as set forth in SEQ ID NO:5, 6, 7, or 8 and a partial nucleotide sequence comprising preferably 15 or more consecutive nucleotides, more preferably 17 or more consecutive nucleotides, and more preferably 20 or more consecutive nucleotides from the 3′ end of the nucleotide sequence as set forth in SEQ ID NO:5, 6, 7, or 8.
  • first polynucleotide “comprises the first nucleotide sequence at the 3′ end” encompasses both a case where the whole nucleotide sequence of the first polynucleotide consists only of the first nucleotide sequence and a case where the first polynucleotide comprises the first nucleotide sequence and a further nucleotide sequence linked to the first nucleotide sequence at the 5′ end thereof.
  • nucleotide sequence does not substantially inhibit the first nucleic acid amplification reaction, and the number of nucleotides thereof is, for example, 20 or fewer nucleotides, preferably 10 or fewer nucleotides, more preferably 5 or fewer nucleotides, and more preferably 1 or 2 nucleotides.
  • the first primers comprising the first polynucleotide consisting of the nucleotide sequences of SEQ ID NOS: 5, 6, 7, and 8 are referred to as SF1, SF2, SF3, and SF4, respectively, and their positions in the nucleotide sequence of SEQ ID NO:1 are shown in FIGS. 7 - 1 and 7 - 2 .
  • the first primer may consist of the first polynucleotide or may comprise the first polynucleotide and useful chemical structures such as a labeling tag, a labeling substance, and immobilization tag added to the first polynucleotide. Such useful chemical structures can be detected.
  • the other primer is preferably a primer comprising a polynucleotide comprising a nucleotide sequence at the 3′ end which is identical or homologous to a partial nucleotide sequence of 10 to 50 consecutive nucleotides, preferably 15 to 30 consecutive nucleotides comprised in the complementary nucleotide sequence to the nucleotide sequence of SEQ ID NO:1, which is complementary to a region positioned more closely to the 3′ end than the first nucleotide in the nucleotide sequence of SEQ ID NO:1, and more preferably the second primer.
  • the second partial nucleotide sequence described in association with the second primer is a partial nucleotide sequence of 10 or more consecutive nucleotides, preferably 15 or more consecutive nucleotides, more preferably 17 or more consecutive nucleotides, more preferably 20 or more consecutive nucleotides, and more preferably 23 or more consecutive nucleotides comprised in the complementary nucleotide sequence to the nucleotide sequence of SEQ ID NO:1.
  • the upper limit of the number of nucleotides in the second partial nucleotide sequence is not particularly limited, and is preferably 50 or fewer nucleotides, more preferably 40 or fewer nucleotides, more preferably 35 or fewer nucleotides, and more preferably 30 or fewer nucleotides.
  • the second partial nucleotide sequence comprises the complementary nucleotide of the second nucleotide as a nucleotide within 2 nucleotides from the 3′ end, preferably as a nucleotide at the 3′ end, a region on the 3′ end side from a nucleotide corresponding to the complementary nucleotide of the second nucleotide in the nucleotide sequence on the antisense chain of gene 1 in the template nucleic acid is specifically elongated from the 3′ end of the second primer in the first nucleic acid amplification reaction.
  • the second polynucleotide comprises the second nucleotide sequence which is identical or homologous to the second partial nucleotide sequence at the 3′ end.
  • the term “homologous” is defined as described above.
  • the second partial nucleotide sequence and the second nucleotide sequence are identical to each other, preferably in a region of 3 nucleotides at the 3′ end, more preferably in a region of 5 nucleotides at the 3′ end, more preferably in a region of 8 nucleotides at the 3′ end, and more preferably in a region of 10 nucleotides at the 3′ end (further preferably in a region of 15 nucleotides at the 3′ end if the length of the second partial nucleotide sequence is 15 or more nucleotides, further preferably in a region of 17 nucleotides at the 3′ end if the length of the second partial nucleotide sequence is 17 or more nucleotides, and further
  • the second nucleotide sequence include the nucleotide sequence as set forth in SEQ ID NO:9, 10, 11, or 12 and a partial nucleotide sequence comprising preferably 15 or more consecutive nucleotides, more preferably 17 or more consecutive nucleotides, and more preferably 20 or more consecutive nucleotides from the 3′ end of the nucleotide sequence as set forth in SEQ ID NO:9, 10, 11, or 12.
  • the expression that the second polynucleotide “comprises the second nucleotide sequence at the 3′ end” encompasses both a case where the whole nucleotide sequence of the second polynucleotide consists only of the second nucleotide sequence and a case where the second polynucleotide comprises the second nucleotide sequence and a further nucleotide sequence linked to the second nucleotide sequence at the 5′ end thereof.
  • nucleotide sequence does not substantially inhibit the first nucleic acid amplification reaction, and the number of nucleotides thereof is, for example, 20 or fewer nucleotides, preferably 10 or fewer nucleotides, more preferably 5 or fewer nucleotides, and more preferably 1 or 2 nucleotides.
  • the second primers comprising the second polynucleotides consisting of the nucleotide sequences of SEQ ID NOS: 9, 10, 11, and 12 are referred to as SR1, SR2, SR3, and SR4, respectively, and their positions in the nucleotide sequence of SEQ ID NO:1 are shown in FIGS. 7 - 1 and 7 - 2 .
  • the second primer may consist of the second polynucleotide or may comprise the second polynucleotide and useful chemical structures such as a labeling tag, a labeling substance, and immobilization tag added to the second polynucleotide. Such useful chemical structures can be detected.
  • the other primer is preferably a primer comprising a polynucleotide comprising a nucleotide sequence at the 3′ end which is identical or homologous to a partial nucleotide sequence of 10 to 50 consecutive nucleotides, preferably 15 to 30 consecutive nucleotides comprised in the nucleotide sequence of SEQ ID NO:1, which is a region positioned more closely to the 5′ end than the second nucleotide in the nucleotide sequence of SEQ ID NO:1, and more preferably the first primer.
  • the first primer set is a combination of the first primer and the second primer, it is sufficient that the first primer and the second primer are selected so that the first nucleotide and the second nucleotide are identical to each other, or the first nucleotide is positioned more closely to the 5′ end than the second nucleotide in the nucleotide sequence of SEQ ID NO:1.
  • the first nucleotide for the first primer is at position 94, 130, 1312, or 1348 in the nucleotide sequence of SEQ ID NO:1
  • the second nucleotide for the second primer is at position 187, 358, 791, or 1467 in SEQ ID NO:1, provided that the first nucleotide is identical to the second nucleotide or is positioned more closely to the 5′ end than the second nucleotide in the nucleotide sequence of SEQ ID NO:1.
  • the second determination step is a step of determining the presence of the nucleotide sequence of SEQ ID NO:2 in a nucleic acid derived from an onion.
  • a nucleotide sequence comprising at least a partial nucleotide sequence of the nucleotide sequence of SEQ ID NO:2 or of a nucleotide sequence derived from SEQ ID NO:2 substitution of T with U or the complementary nucleotide sequence to the partial nucleotide sequence is detected in the nucleic acid derived from the onion.
  • the second determination step it is preferable to determine the presence of a second mutation site comprising one or more nucleotides selected from nucleotides at positions 34, 70, 1667, 1703, 127, 409, 943, and 1822 in the nucleotide sequence of SEQ ID NO:2.
  • a second mutation site comprising one or more nucleotides selected from nucleotides at positions 34, 70, 1667, 1703, 127, 409, 943, and 1822 in the nucleotide sequence of SEQ ID NO:2.
  • a second nucleic acid amplification reaction As a specific means for determining the presence of the second mutation site in the nucleotide sequence of SEQ ID NO:2 in the nucleic acid derived from the onion, it is preferable to perform a second nucleic acid amplification reaction using the following second primer set and a nucleic acid derived from an onion, in particular, a genomic DNA as a template and to confirm an amplification product of the second nucleic acid amplification reaction.
  • a second primer set preferably comprises at least either one of
  • a third primer comprising a polynucleotide comprising a nucleotide sequence which is identical or homologous to a partial nucleotide sequence comprising the second mutation site in the nucleotide sequence of SEQ ID NO:2, and
  • a fourth primer comprising a polynucleotide comprising a nucleotide sequence which is identical or homologous to the complementary nucleotide sequence to the partial nucleotide sequence comprising the second mutation site in the nucleotide sequence of SEQ ID NO:2, and particularly preferably both the third and fourth primers.
  • Examples of a preferred embodiment of the third primer include a third primer comprising a third polynucleotide comprising a third nucleotide sequence at the 3′ end which is identical or homologous to a third partial nucleotide sequence of 10 or more consecutive nucleotides comprised in the nucleotide sequence of SEQ ID NO:2 comprising a third nucleotide selected from positions 34, 70, 1667, 1703, 127, 409, 943, and 1822 in the nucleotide sequence of SEQ ID NO:2 within 2 nucleotides from the 3′ end.
  • Examples of a preferred embodiment of the fourth primer include a fourth primer comprising a fourth polynucleotide comprising a fourth nucleotide sequence at the 3′ end which is identical or homologous to a fourth partial nucleotide sequence of 10 or more consecutive nucleotides comprised in the complementary nucleotide sequence to the nucleotide sequence of SEQ ID NO:2 comprising a nucleotide complementary to a fourth nucleotide selected from positions 34, 70, 1667, 1703, 127, 409, 943, and 1822 in the nucleotide sequence of SEQ ID NO:2 within 2 nucleotides from the 3′ end.
  • the third partial nucleotide sequence described in association with the third primer is a partial nucleotide sequence of 10 or more consecutive nucleotides, preferably 15 or more consecutive nucleotides, more preferably 17 or more consecutive nucleotides, more preferably 20 or more consecutive nucleotides, and more preferably 23 or more consecutive nucleotides comprised in the nucleotide sequence of SEQ ID NO:2.
  • the upper limit of the number of nucleotides in the third partial nucleotide sequence is not particularly limited, and is preferably 50 or fewer nucleotides, more preferably 40 or fewer nucleotides, more preferably 35 or fewer nucleotides, and more preferably 30 or fewer nucleotides.
  • the third partial nucleotide sequence comprises the third nucleotide as a nucleotide within 2 nucleotides from the 3′ end, preferably as a nucleotide at the 3′ end, a region on the 3′ end side from a nucleotide corresponding to the third nucleotide in the nucleotide sequence on the sense chain of gene 2 in the template nucleic acid is specifically elongated from the 3′ end of the third primer in the second nucleic acid amplification reaction.
  • the third polynucleotide comprises the third nucleotide sequence which is identical or homologous to the third partial nucleotide sequence at the 3′ end.
  • the term “homologous” is defined as described above.
  • the third partial nucleotide sequence and the third nucleotide sequence are identical to each other, preferably in a region of 3 nucleotides at the 3′ end, more preferably in a region of 5 nucleotides at the 3′ end, more preferably in a region of 8 nucleotides at the 3′ end, and more preferably in a region of 10 nucleotides at the 3′ end (further preferably in a region of 15 nucleotides at the 3′ end if the length of the third partial nucleotide sequence is 15 or more nucleotides, further preferably in a region of 17 nucleotides at the 3′ end if the length of the third partial nucleotide sequence is 17 or more nucleotides, and further
  • the third nucleotide sequence include the nucleotide sequence as set forth in SEQ ID NO:13, 14, 15, or 16 and a partial nucleotide sequence comprising preferably 15 or more consecutive nucleotides, more preferably 17 or more consecutive nucleotides, and more preferably 20 or more consecutive nucleotides from the 3′ end of the nucleotide sequence as set forth in SEQ ID NO:13, 14, 15, or 16.
  • the expression that the third polynucleotide “comprises the third nucleotide sequence at the 3′ end” encompasses both a case where the whole nucleotide sequence of the third polynucleotide consists only of the third nucleotide sequence and a case where the third polynucleotide comprises the third nucleotide sequence and a further nucleotide sequence linked to the third nucleotide sequence at the 5′ end thereof.
  • nucleotide sequence does not substantially inhibit the second nucleic acid amplification reaction, and the number of nucleotides thereof is, for example, 20 or fewer nucleotides, preferably 10 or fewer nucleotides, more preferably 5 or fewer nucleotides, and more preferably 1 or 2 nucleotides.
  • the third primers comprising the third polynucleotide consisting of the nucleotide sequences of SEQ ID NOS: 13, 14, 15, and 16 are referred to as UF1, UF2, UF3, and UF4, respectively, and their positions in the nucleotide sequence of SEQ ID NO:2 are shown in FIGS. 8 - 1 and 8 - 2 .
  • the third primer may consist of the third polynucleotide or may comprise the third polynucleotide and useful chemical structures such as a labeling tag, a labeling substance, and immobilization tag added to the third polynucleotide. Such useful chemical structures can be detected.
  • the other primer is preferably a primer comprising a polynucleotide comprising a nucleotide sequence at the 3′ end which is identical or homologous to a partial nucleotide sequence of 10 to 50 consecutive nucleotides, preferably 15 to 30 consecutive nucleotides comprised in the complementary nucleotide sequence to the nucleotide sequence of SEQ ID NO:2 which is complementary to a region positioned more closely to the 3′ end than the third nucleotide in the nucleotide sequence of SEQ ID NO:2, and more preferably the fourth primer.
  • the fourth partial nucleotide sequence described in association with the fourth primer is a partial nucleotide sequence of 10 or more consecutive nucleotides, preferably 15 or more consecutive nucleotides, more preferably 17 or more consecutive nucleotides, more preferably 20 or more consecutive nucleotides, and more preferably 23 or more consecutive nucleotides comprised in the complementary nucleotide sequence to the nucleotide sequence of SEQ ID NO:2.
  • the upper limit of the number of nucleotides in the fourth partial nucleotide sequence is not particularly limited, and is preferably 50 or fewer nucleotides, more preferably 40 or fewer nucleotides, more preferably 35 or fewer nucleotides, and more preferably 30 or fewer nucleotides.
  • the fourth partial nucleotide sequence comprises the complementary nucleotide of the fourth nucleotide as a nucleotide within 2 nucleotides from the 3′ end, preferably as a nucleotide at the 3′ end, and a region on the 3′ end side from a nucleotide corresponding to the complementary nucleotide of the fourth nucleotide in the nucleotide sequence on the antisense chain of gene 2 in the template nucleic acid is specifically elongated from the 3′ end of the fourth primer in the second nucleic acid amplification reaction.
  • the fourth polynucleotide comprises the fourth nucleotide sequence which is identical or homologous to the fourth partial nucleotide sequence at the 3′ end.
  • the term “homologous” is defined as described above.
  • the fourth partial nucleotide sequence and the fourth nucleotide sequence are identical to each other, preferably in a region of 3 nucleotides at the 3′ end, more preferably in a region of 5 nucleotides at the 3′ end, more preferably in a region of 8 nucleotides at the 3′ end, and more preferably in a region of 10 nucleotides at the 3′ end (further preferably in a region of 15 nucleotides at the 3′ end if the length of the fourth partial nucleotide sequence is 15 or more nucleotides, further preferably in a region of 17 nucleotides at the 3′ end if the length of the fourth partial nucleotide sequence is 17 or more nucleotides, and further
  • the fourth nucleotide sequence include the nucleotide sequence as set forth in SEQ ID NO:17, 18, 19, or 20 and a partial nucleotide sequence comprising preferably 15 or more consecutive nucleotides, more preferably 17 or more consecutive nucleotides, and more preferably 20 or more consecutive nucleotides from the 3′ end of the nucleotide sequence as set forth in SEQ ID NO:17, 18, 19, or 20.
  • the fourth polynucleotide “comprises the fourth nucleotide sequence at the 3′ end” encompasses both a case where the whole nucleotide sequence of the fourth polynucleotide consists only of the fourth nucleotide sequence and a case where the fourth polynucleotide comprises the fourth nucleotide sequence and a further nucleotide sequence linked to the fourth nucleotide sequence at the 5′ end thereof.
  • nucleotide sequence does not substantially inhibit the second nucleic acid amplification reaction, and the number of nucleotides thereof is, for example, 20 or fewer nucleotides, preferably 10 or fewer nucleotides, more preferably 5 or fewer nucleotides, and more preferably 1 or 2 nucleotides.
  • the fourth primers comprising the fourth polynucleotide consisting of the nucleotide sequence of SEQ ID NOS: 17, 18, 19, and 20 are referred to as UR1, UR2, UR3, and UR4, respectively, and their positions in the nucleotide sequence of SEQ ID NO:2 are shown in FIGS. 8 - 1 and 8 - 2 .
  • the fourth primer may consist of the fourth polynucleotide or may comprise the fourth polynucleotide and useful chemical structures such as a labeling tag, a labeling substance, and immobilization tag added to the fourth polynucleotide. Such useful chemical structures can be detected.
  • the other primer is preferably a primer comprising a polynucleotide comprising a nucleotide sequence at the 3′ end which is identical or homologous to a partial nucleotide sequence of 10 to 50 consecutive nucleotides, preferably 15 to 30 consecutive nucleotides comprised in the nucleotide sequence of SEQ ID NO:2, which is a region positioned more closely to the 5′ end than the fourth nucleotide in the nucleotide sequence of SEQ ID NO:2, and more preferably the third primer.
  • the second primer set is a combination of the third primer and the fourth primer, it is sufficient that the third primer and the fourth primer are selected so that the third nucleotide and the fourth nucleotide are identical to each other, or the third nucleotide is positioned more closely to the 5′ end than the fourth nucleotide in the nucleotide sequence of SEQ ID NO:2.
  • the third nucleotide for the third primer is at position 34, 70, 1667, or 1703 in the nucleotide sequence of SEQ ID NO:2
  • the fourth nucleotide for the fourth primer is at position 127, 409, 943, or 1822 in SEQ ID NO:2, provided that the third nucleotide is identical to the fourth nucleotide or is positioned more closely to the 5′ end than the fourth nucleotide in the nucleotide sequence of SEQ ID NO:2.
  • both the first nucleic acid amplification reaction and the second nucleic acid amplification reaction can be performed according to a usual polymerase chain reaction (PCR) method.
  • PCR polymerase chain reaction
  • a nucleic acid, such as a genomic DNA or a cDNA, used as a template can be prepared from an onion according to a usual method.
  • DNA polymerase used for PCR is a thermostable DNA polymerase
  • DNA polymerases are not particularly limited. In the present invention, commercially available DNA polymerases can be used. Additionally, primer concentration, number of cycles, temperature, time, compositions of buffer solutions, and other conditions can be appropriately selected depending on the DNA polymerase used, the concentration of each primer, and other conditions.
  • a polynucleotide fragment comprising a predetermined target region is produced as an amplification product when the nucleic acid derived from the target onion contains a nucleotide sequence to be detected.
  • the method of confirming the amplification product of the nucleic acid amplification reaction is not particularly limited, and examples thereof include a method in which, after completion of the nucleic acid amplification reaction, the reaction mixture of the nucleic acid amplification reaction is fractioned by gel electrophoresis, and the presence of a band of the size corresponding to the polynucleotide fragment comprising the predetermined target region is confirmed and a method in which the amplification product is labelled and detected. Additionally, the amplification product can also be detected by a real-time PCR method while performing the nucleic acid amplification reaction.
  • the method of discriminating traits of an onion of the present invention is a method comprising
  • the first determination step comprising determining presence of a first mutation site comprising one or more nucleotides selected from nucleotides at positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleotide sequence of SEQ ID NO:1 in the nucleic acid, wherein
  • the onion is discriminated to be an onion with no pungent taste and/or tear-inducing property if the presence of the nucleotide sequence of SEQ ID NO:1 is not determined in the first determination step;
  • the onion is discriminated to be an onion with a pungent taste and/or a tear-inducing property if the presence of the nucleotide sequence of SEQ ID NO:1 is determined in the first determination step.
  • This method is the “second discrimination method” of the present invention.
  • the first determination step in the second discrimination method is the same as the first determination step in the first discrimination method.
  • Specific embodiments of the first determination step in the second discrimination method are the same as the specific embodiments of the first determination step in the first discrimination method.
  • the first determination step in the second discrimination method preferably comprises performing a first nucleic acid amplification reaction using a first primer set comprising at least either one of a first primer and a second primer and a nucleic acid of an onion as a template and confirming an amplification product of the first nucleic acid amplification reaction.
  • the first primer, the second primer, the first primer set, the first nucleic acid amplification reaction, and the method of detecting the amplification product in this embodiment of the second discrimination method are as explained for the first discrimination method.
  • the first or second discrimination method of the present invention may be used in combination with further methods for discriminating an onion with no pungent taste and/or tear-inducing property.
  • an onion with no pungent taste and/or tear-inducing property can be discriminated accurately.
  • Examples of further methods for discriminating an onion with no pungent taste and/or tear-inducing property include discriminating whether the onion is an onion with no pungent taste and/or tear-inducing property based on one or more of the following traits as indicator(s):
  • An onion having one or more of the above traits can be discriminated to be an onion with no pungent taste and/or tear-inducing property.
  • the present invention also provides
  • an onion with no pungent taste and/or tear-inducing property can be accurately selected among onions produced by crossing using the first or second discrimination method of the present invention.
  • the selected onion with no pungent taste and/or tear-inducing property can be used for further breeding.
  • Both the first primer and the second primer are useful as a primer to specifically amplify a part comprising a nucleotide sequence corresponding to the nucleotide sequence of SEQ ID NO:1 in a nucleic acid derived from an onion.
  • the first primer set comprising the first primer and the second primer in which the first nucleotide and the second nucleotide are identical to each other, or the first nucleotide is positioned more closely to the 5′ end than the second nucleotide in the nucleotide sequence of SEQ ID NO:1 is useful as a primer set to specifically amplify a part comprising a nucleotide sequence corresponding to the nucleotide sequence of SEQ ID NO:1 in the nucleic acid derived from the onion.
  • Both the third primer and the fourth primer are useful as a primer to specifically amplify a part comprising a nucleotide sequence corresponding to the nucleotide sequence of SEQ ID NO:2 in a nucleic acid derived from an onion.
  • the second primer set comprising the third primer and the fourth primer in which the third nucleotide and the fourth nucleotide are identical to each other, or the third nucleotide is positioned more closely to the 5′ end than the fourth nucleotide in the nucleotide sequence of SEQ ID NO:2 is useful as a primer set to specifically amplify a part comprising a nucleotide sequence corresponding to the nucleotide sequence of SEQ ID NO:2 in a nucleic acid derived from an onion.
  • kits comprising one or more of these primers or primer sets is useful as a kit to discriminate traits of an onion.
  • the kit can further comprise various components (e.g., DNA polymerase, various buffer solutions, dNTPs) used to perform a nucleic acid amplification reaction and confirm an amplification product.
  • Alliinase gene 1 for which the cDNA nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:1, is not present in an onion with no pungent taste and/or tear-inducing property and is present in a usual onion with a pungent taste and/or a tear-inducing property. Therefore, alliinase gene 1 is useful as a marker gene to detect a pungent taste and/or a tear-inducing property of an onion. If alliinase gene 1 is detected in a nucleic acid derived from an onion, the onion can be discriminated to be an onion with a pungent taste and/or a tear-inducing property. If alliinase gene 1 is not detected in a nucleic acid derived from an onion, the onion is discriminated to be an onion with no pungent taste and/or tear-inducing property.
  • the present invention further includes the following inventions A to C:
  • a method of manufacturing a plant body of an onion with no pungent taste and/or tear-inducing property, a progeny thereof, or a part thereof comprising the following steps: (i) a step of inducing a mutation in an onion seed; (ii) a step of cultivating the onion seed with the induced mutation to obtain a plant body of the onion or a part thereof, and (iii) a step of discriminating and selecting the plant body of an onion with no pungent taste and/or tear-inducing property or a part thereof by the first or second discrimination method of the present invention from plant bodies or parts thereof of the obtained onions.
  • a method of manufacturing a plant body of an onion with no pungent taste and/or tear-inducing property, a progeny thereof, or a part thereof, comprising crossing a first onion discriminated to be an onion with no pungent taste and/or tear-inducing property by the first or second discrimination method of the present invention and a second onion.
  • Patent Literature 1 may be referred to for specific aspects of the inventions A to C.
  • Example 1 Acquiring the Gene Sequence of Alliinase Associated with a Pachet Taste and/or a Tear-Inducing Property of an Onion
  • Analytical method paired end sequencing; number of specimens, 6; number of lanes, 3; read nucleotide length, 100 nucleotides/read
  • Chastity is represented by a formula of “I1/(I1+I2),” where the largest value of signals from 4 different nucleotides is I1 and the second largest value is I2. In this example, data were selected under a condition of “I1/(I1+I2)>0.6.”
  • the selected data were sorted out by specimen according to the specimen unique index information.
  • the assembly data were annotated by BLAST search.
  • the BLASTX was used as the annotation program, and the amino acid sequences in RefSeq-Fungi, RefSeq-Microbial, RefSeq-Plant of NCBI and the amino acid sequences and the gene sequences (converted to amino acid sequence) registered under Allieae in the NCBI classification were integrated and used as the database.
  • the BLASTX parameter used was evalue 1E-5/num_alignments 100/outfmt “6 qseqid sseqid pident length mismatch gapopen qstart qend sstart send evalue bitscore qlen slen stitle qcovs qcovhsp”/, and default conditions were used for others.
  • a cDNA nucleotide sequence encoding alliinase comprising “amino acid sequence 5” disclosed in Patent Literature 1 was acquired.
  • the cDNA nucleotide sequence of “amino acid sequence 5” encoding alliinase is shown as SEQ ID NO:1 in the sequence listing.
  • a forward primer corresponding to the end part of the nucleotide sequence of SEQ ID NO:1 encoding alliinase that comprises the start codon and a reverse primer corresponding to the end part of the nucleotide sequence that comprises the stop codon were prepared, 100 mg each of samples were collected from both onion #6 and the control variety used in Example 1, the genomic DNA was prepared using the DNeasy Plant Mini Kit (manufactured by Qiagen) according to the attached manual, and a polymerase chain reaction (PCR) was performed using the prepared genomic DNA as a template. Amplification products were analyzed using the MultiNA Electrophoresis System (manufactured by Shimadzu Corporation). The nucleotide sequences of the primers are as shown below, and the positions of the primers are shown in FIGS. 1 - 1 , 1 - 2 , 1 - 3 , and 1 - 4 .
  • Forward primer 1 (SEQ ID NO: 3) 5′-ATGGAGTCTTACCACAAAGTTGGCAGT-3′
  • Reverse primer 2 (SEQ ID NO: 4) 5′-GTAGCCCATACATGATCACAAACATGAAC-3′
  • FIG. 2 The results of the analysis of the amplification products by electrophoresis are shown in FIG. 2 . Amplification products of the same size were confirmed for both onion #6 and the control variety ( FIG. 2 ). The result of directly sequencing the amplification products confirmed that the product of amplification of the genomic DNA of onion #6 as a template had the genomic DNA nucleotide sequence as set forth in SEQ ID NO:2 exhibiting an approximately 95% identity to the nucleotide sequence of SEQ ID NO:1 in the exon parts (Table 1).
  • the first exon is at positions 50 to 339
  • the second exon is at positions 340 to 651
  • the third exon is at positions 652 to 935
  • the fourth exon is at positions 936 to 1235
  • the fifth exon is at positions 1236 to 1621.
  • the part corresponding to the first exon in SEQ ID NO:1 is positions 1 to 279
  • the part corresponding to the second exon in SEQ ID NO:1 is positions 391 to 702
  • the part corresponding to the third exon in SEQ ID NO:1 is positions 804 to 1087
  • the part corresponding to the fourth exon in SEQ ID NO:1 is positions 1188 to 1487
  • the part corresponding to the fifth exon in SEQ ID NO:1 is positions 1591 to 1975.
  • the product of amplification of the genomic DNA of the control variety onion as a template comprised a mixture of the amplification product comprising of genomic DNA fragments of the gene for which the cDNA nucleotide sequence is the nucleotide sequence of SEQ ID NO:1 and the amplification product comprising genomic DNA fragments comprising the nucleotide sequence of SEQ ID NO:2, and clear results could not be obtained by direct sequencing.
  • alliinase gene 1 both the alliinase gene for which the cDNA nucleotide sequence comprised the nucleotide sequence of SEQ ID NO:1 (hereinafter referred to as “alliinase gene 1”) and the alliinase gene for which the genomic DNA nucleotide sequence comprised the nucleotide sequence of SEQ ID NO:2 (hereinafter referred to as “alliinase gene 2”) and that onion #6 had alliinase gene 2 but did not have alliinase gene 1 as alliinase genes.
  • alliinase gene 2 appears to be a pseudogene, which is not expressed, because RNA corresponding to this gene was not detected in the RNA-seq analysis of Example 1.
  • primers to detect alliinase gene 1 and alliinase gene 2 specifically the nucleotide sequences of the alliinase genes of SEQ ID NO:1, SEQ ID NO:2, and GenBank Accession No. AAA32639.1 were compared, and primers that specifically amplified only either one of alliinase gene 1 and alliinase gene 2 were prepared, wherein one or more nucleotides at the 3′ end of each primer are identical to only either one of the nucleotide sequence of SEQ ID NO:1 and the nucleotide sequence of SEQ ID NO:2 at the corresponding positions.
  • primers were prepared for a part common to the nucleotide sequence of SEQ ID NO:1 and the nucleotide sequence of SEQ ID NO:2.
  • the nucleotide sequence of each primer is shown in Table 2, and the positions of the primers are shown in FIGS. 7 - 1 , 7 - 2 , 8 - 1 , and 8 - 2 .
  • a mixture of UF3 primers (SEQ ID NO:15) that comprised T or C at the position of Y were used.
  • PCR was performed on the genomic DNAs of onion #6 and the control variety prepared in Example 2 as templates using the prepared primers, and amplification products were analyzed using the MultiNA Electrophoresis System (manufactured by Shimadzu Corporation). The results are shown in FIGS. 3 - 1 and 3 - 2 . Combinations of the forward primer and the reverse primer used are shown in FIGS. 3 - 1 and 3 - 2 .
  • the effectiveness of the above-described primers was investigated using the genomic DNAs of 14 commercially available onion varieties shown in Table 2, the above-described control variety, and onion #6 as templates.
  • the conditions for PCR were the same as the above-described conditions.
  • the results are shown in FIG. 4 .
  • the lane numbers in FIG. 4 correspond to the onion variety numbers in Table 3.
  • Lanes 1 to 14 show the analysis results for commercially available varieties
  • lane 15 is the analysis results for the above-described control variety
  • lane 16 is the analysis results for onion #6.
  • the results shown in FIG. 4 confirmed that the 14 commercially available varieties and the control variety had alliinase gene 1 and alliinase gene 2, and that onion #6 had alliinase gene 2 but did not have alliinase gene 1.
  • the above-described primers can be used to select a tearless and non-pungent onion which is incorporated with useful traits from progenies obtained by crossing the tearless and non-pungent onion #6 and a second onion having the useful traits by a common crossing method.
  • a long-day onion having a good bulb size was crossed with onion #6.
  • onion #6 and a male sterile long-day onion having a trait of a good bulb size were crossed by a conventional method in Hokkaido to obtain F1 seeds.
  • the F1 seeds were seeded, and bulbs of the F1 onions (F1 bulbs) were harvested by the conventional planting method in Hokkaido.
  • the harvested F1 bulbs were self-reproduced and cultivated to obtain F2 seeds.
  • the obtained F2 seeds were cultivated by the conventional method to obtain bulbs of the F2 onions (F2 bulbs).
  • Onion selection was performed by sampling leaves of 4249 bulbs in the process of cultivating the F2 bulbs, extracting genomic DNAs, and performing PCR using the genomic DNAs as templates and three primer sets of SF2-SR1, SF1-SR1, and UF2-UR1 among the above-described primers. Then, F2 bulbs were selected for which amplification products corresponding to alliinase gene 2 were obtained, and amplification products corresponding to alliinase gene 1 were not obtained. The F2 bulbs selected according to this criterion are presumed to be tearless and non-pungent onion bulbs. As a result, 866 onion bulbs were selected from 4249 bulbs.
  • FIG. 5 shows one example of the detection results for the amplification products of PCR using genomic DNAs from leaves of a plurality of F2 onions as templates and SF2-SR1 as a primer set. The upper column of FIG. 5 shows the names of the F2 onion samples.
  • the present invention can be used to discriminate an onion with no pungent taste in the fields such as agriculture and food manufacturing.

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